Rickettsia felis was originally identified in the United States as a human pathogen in 1991; and, with increased recognition worldwide, it is now realized that human infections caused by R. felis account for up to 6% of the causes of fever among non-malarial conditions in sub-Saharan Africa. Our long-term goal is to elucidate the molecular mechanisms that are critical to rickettsial transmission by fleas in order to better understand the epidemiology of flea-borne rickettsial diseases and identify points of intervention. In addition to being a horizontally transmitted flea-borne pathogen, R. felis is alsoa vertically maintained endosymbiont of non-hematophagous insects. For arthropod-borne Rickettsia spp., the level of virulence is expected to be governed by the relative importance of vertical and horizontal transmission for maintenance of the rickettsiae. Therefore, the central hypothesis being tested is that there are host-dependent mechanisms that are essential to rickettsial virulence and transmission. The mechanism by which R. felis differentiates the host environment and executes either horizontal or vertical routes for successful transmission and maintenance in arthropod populations are not known. The experimental focus of this proposal is to delineate the Rickettsia- and arthropod-dependent factors contributing to successful horizontal transmission through comparative and functional analyses of R. felis in different arthropod hosts, and at the arthropod-host interface. The biological and molecular events underlying the transmission of R. felis in flea hosts are poorly defined and are critical to clearl understanding the epidemiology of flea-borne rickettsial diseases and identify points of intervention. The previously undefined parameters of rickettsial infection in fleas will be determined in the following specific aims Aim 1. Characterization of rickettsial transcript expression profile in cat fleas compared to non- hematophagous arthropods. Using RNA-seq, expressed rickettsial transcripts will be identified in R. felis- infected fleas, during flea hematophagy, and in rickettsiae deposited into blood during horizontal transmission between cofeeding fleas. Comparisons will be made to constitutively infected booklice. Subsequently, unique rickettsial transcripts will be functionally characterized via targeted gene disruption and their requirement for horizontal transmission assessed. Utilizing in vivo systems, we will be able to test the hypothesis that R. felis has an essential transcript profile necessary for horizontal transmission during flea bloodmeal acquisition. Aim 2. Determination of cat flea-derived salivary gland secreted factors essential for horizontal transmission of Rickettsia. The salivary gland transcription profile has been initially characterized in cat fleas and salivary gland transcripts identified as secreted molecules will be assessed in R. felis-infected cat fleas during the feeding period. Differentially expressed transcripts associated with R. felis infection of fleas will be functionally characterized by RNA-mediated interference and the necessity of these flea- derived factors for horizontal transmission assessed. Utilizing a horizontal transmission bioassay, the hypothesis that flea-derived factors are essential to successful horizontal transmission of R. felis will be tested.